Fast photochemical method of labelling nucleic acids for detection purposes in hybridization assays

ABSTRACT

A labeled nucleic acid probe comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component, and (c) a label chemically linked to the nucleic acid-binding ligand. The label can be a specifically bindable ligand such as a hapten or biotin, an enzyme such as a  beta -galactosidase or horse radish peroxidase, a fluorescent radical, a phycobiliprotein, a luminescent radical, or a radioisotope. The probe can be used in assays of nucleic acids, taking advantage of the ability of the nucleic acid component to hybridize.

This is a continuation-in-part of application Ser. No. 513,932, filedJuly 14, 1983, now abandoned.

The present invention relates to a photochemical method of labellingnucleic acids for detection purposes in hybridization assays for thedetermination of specific polynucleotide sequences.

The most efficient and sensitive method of detection of nucleic acidssuch as DNA after hybridization requires radioactively labelled DNA. Theuse of autoradiography and enzymes makes the assay time consuming andrequires experienced technical people. Recently, a non-radioactivemethod of labelling DNA has been described by Ward et al, European Pat.Appl. No. 63,879; they use the method of nick translation to introducebiotinylated U residue to DNA replacing T. The biotin residue is thenassayed with antibiotin antibody or an avidin containing system. Thedetection in this case is quicker than autoradiography but the method ofnick translation is a highly skilled art. Moreover, biotinylation usingbiotinylated UTP works only for thymine-containing polynucleotides. Useof other nucleotide triphosphates is very difficult because the chemicalderivatization of A or G or C (containing --NH₂) with biotin requireselaborate and highly skilled organic chemists.

It is accordingly an object of the present invention to provide asimplified system for detection of nucleic acids by hybridizationassays, which system can be easily produced and used without thedisadvantages noted hereinabove.

These and other objects and advantages are realized in accordance withthe present invention pursuant to which the nucleic acid is labeled bymeans of photochemistry, employing a photoreactive nucleic acid-bindingligand, e.g., an intercalator compound such as a furocoumarin or aphenanthridine compound or a non-intercalator compound such asnetropsin, distamycin, Hoechst 33258 and bis-benzimidazole to link thenucleic acid to a label which can be "read" or assayed in conventionalmanner, including fluorescence detection. The end product is thus alabeled nucleic acid probe comprising (a) a nucleic acid component, (b)an intercalator or other nucleic acid-binding ligand photochemicallylinked to the nucleic acid component, and (c) a label chemically linkedto (c).

The novel photochemical method provides more favorable reactionconditions than the usual chemical coupling method for biochemicallysensitive substances. By using proper wavelengths for irradiation, DNA,RNA and proteins can be modified without affecting the native structureof the polymers. The nucleic acid-binding ligand, hereinafterexemplified by an intercalator, and label can first be coupled and thenphotoreacted with the nucleic acid or the nucleic acid can first bephotoreacted with the intercalator and then coupled to the label. Ageneral scheme for coupling a nucleic acid, exemplified bydouble-stranded DNA, to a label such as a hapten or enzyme is asfollows: ##STR1##

Where the hybridizable portion of the probe is in a double strandedform, such portion is then denatured to yield a hybridizable singlestranded portion. Alternatively, where the labeled DNA comprises thehybridizable portion already in single stranded form, suchdenaturization can be avoided if desired. Alternatively, double strandedDNA can be labeled by the approach of the present invention afterhybridization has occurred using a hybridization format which generatesdouble stranded DNA only in the presence of the sequence to be detected.

To produce specific and efficient photochemical products, it isdesirable that the nucleic acid component and the photoreactiveintercalator compound be allowed to react in the dark in a specificmanner.

For coupling to DNA, aminomethyl psoralen, aminomethyl angelicin andamino alkyl ethidium or methidium azides are particularly usefulcompounds. They bind to double-stranded DNA and only the complexproduces photoadduct. In the case where labeled double-stranded DNA mustbe denatured in order to yield a hybridizable single stranded region,conditions are employed so that simultaneous interaction of two strandsof DNA with a single photoadduct is prevented. It is necessary that thefrequency of modification along a hybridizable single stranded portionof the probe not be so great as to substantially prevent hybridization,and accordingly there preferably will be not more than one site ofmodification per 25, more usually 50, and preferably 100, nucleotidebases. Angelicin derivatives are superior to psoralen compounds formonoadduct formation. If a single-stranded probe is covalently attachedto some extra double-stranded DNA, use of phenanthridium and psoralencompounds is desirable since these compounds interact specifically todouble-stranded DNA in the drak. The chemistry for the synthesis of thecoupled reagents to modify nucleic acids for labelling, described morefully hereinbelow, is similar for all cases.

The nucleic acid component can be singly or doubly stranded DNA or RNAor fragments thereof such as are produced by restriction enzymes or evenrelatively short oligomers.

The nucleic acid-binding ligands of the present invention used to linkthe nucleic acid component to the label can be any suitablephotoreactive form of known nucleic acid-binding ligands. Particularlypreferred nucleic acid-binding ligands are intercalator compounds suchas the furocoumarins, e.g., angelicin (isopsoralen) or psoralen orderivatives thereof which photochemically will react with nucleic acids,e.g., 4'-aminomethyl-4,5'-dimethyl angelicin,4'-aminomethyltrioxsalen(4'-aminomethyl-4,5',8-trimethyl-psoralen,3-carboxy-5- or -8-amino- or -hydroxy-psoralen, as well as mono- orbis-azido aminoalkyl methidium or ethidium compounds. Photoreactiveforms of a variety of other intercalating agents can also be used asexemplified in the following table:

    ______________________________________                                        Intercalator Classes and                                                      Representative Compounds                                                                          Literature References                                     ______________________________________                                        A.   Acridine dyes      Lerman, J. Mol. Biol.                                      proflavin, acridine                                                                              3:18(1961); Bloomfield                                     orange, quinacrine,                                                                              et al, "Physical                                           acriflavine        Chemistry of Nucleic                                                          Acids", Chapter 7, pp.                                                        429-476, Harper and                                                           Rowe, NY(1974)                                                                Miller et al, Bio-                                                            polymers 19:2091(1980)                                B.   Phenanthridines    Bloomfield et al, supra                                    ethidium           Miller et al, supra                                        coralyne           Wilson et al, J. Med.                                                         Chem. 19:1261(1976)                                        ellipticine, ellipticine                                                                         Festy et al, FEBS                                          cation and derivatives                                                                           Letters 17:321(1971);                                                         Kohn et al, Cancer Res.                                                       35:71(1976); LePacq et                                                        al, PNAS (USA)71:                                                             5078(1974); Pelaprat et                                                       al, J. Med. Chem.                                                             23:1330(1980)                                         C.   Phenazines         Bloomfield et al, supra                                    5-methylphenazine cation                                                 D.   Phenothiazines       "                                                        chlopromazine                                                            E.   Quinolines           "                                                        chloroquine                                                                   quinine                                                                  F.   Aflatoxin            "                                                   G.   Polycyclic hydrocarbons                                                                            "                                                        and their oxirane                                                             derivatives                                                                   3,4-benzpyrene     Yang et al, Biochem.                                       benzopyrene diol   Biophys. Res. Comm.                                        epoxide, 1-pyrenyl-                                                                              82:929(1978)                                               oxirane                                                                       benzanthracene-5,6-oxide                                                                         Amea et al, Science                                                           176:47(1972)                                          H.   Actinomycins       Bloomfield et al, supra                                    actinomycin D                                                            I.   Anthracyclinones     "                                                   rhodomycin A                                                                       daunamycin                                                               J.   Thiaxanthenones      "                                                        miracil D                                                                K.   Anthramycin          "                                                   L.   Mitomycin          Ogawa et al, Nucl.                                                            Acids Res., Spec.                                                             Publ. 3:79(1977);                                                             Akhtar et al, Can. J.                                                         Chem. 53:2891(2975)                                   M.   Platinium Complexes                                                                              Lippard, Accts. Chem.                                                         Res. 11:211(1978)                                     N.   Polyintercalators  Waring et al, Nature                                       echinomycin        252:653(1974);                                                                Wakelin, Biochem. J.                                                          157:721(1976)                                              quinomycin         Lee et al, Biochem. J.                                     triostin           173:115(1978): Huang                                       BBM928A            et al, Biochem. 19:                                        tandem             5537(1980): Viswamitra                                                        et al, Nature 289:                                                            817(1981)                                                  diacridines        LePecq et al, PNAS                                                            (USA)72:2915(1975):                                                           Carrellakis et al,                                                            Biochim. Biophys.                                                             Acta 418:277(1976);                                                           Wakelin et al, Biochem                                                        17:5057(1978); Wakelin                                                        et al, FEBS Lett.                                                             104:261(1979); Capelle                                                        et al, Biochem. 18:3354                                                       (1979); Wright et al,                                                         Biochem. 19:5825(1980);                                                       Bernier et al, Biochem.                                                       J. 199:479 (1981); King                                                       et al, Biochem. 21:4982                                                       (1982)                                                     ethidium dimer     Gaugain et al, Biochem.                                                       17:5078(1978); Kuhlman                                                        et al, Nucl. Acids Res.                                                       5:2629(1978); Marlcovits                                                      et al, Anal. Biochem.                                                         94:259(1979): Dervan et                                                       al, JACS 100:1968(1978);                                                      ibid 101:3664(1979).                                       ellipticene dimers Debarre et al, Compt.                                      and analogs        Rend. Ser. D. 284:                                                            81(1977); Pelaprat et                                                         al, J. Med. Chem.                                                             23:1336(1980)                                              heterodimers       Cain et al, J. Med.                                                           Chem. 21:658(1978);                                                           Gaugain et al, Biochem.                                                       17:5078(1978)                                              trimers            Hansen et al, JCS                                                             Chem. Comm. 162(1983);                                                        Atnell et al, JACS                                                            105:2913(1983)                                        O.   Norphillin A       Loun et al, JACS 104:                                                         3213(1982)                                            P.   Fluorenes and fluorenones                                                                        Bloomfield et al, supra                                    fluorenodiamines   Witkowski et al,                                                              Wiss. Beitr.-Martin-                                                          Luther-Univ. Halle                                                            Wittenberg, 11(1981)                                  Q.   Furocoumarins                                                                 angelicin          Venema et al, MGG,                                                            Mol. Gen. Genet.                                                              179;1 (1980)                                               4,5'-dimethylangelicin                                                                           Vedaldi et al, Chem.-                                                         Biol. Interact. 36:                                                           275(1981)                                                  psoralen           Marciani et al, Z.                                                            Naturforsch B 27(2):                                                          196(1972)                                                  8-methoxypsoralen  Belognzov et al, Mutat.                                                       Res. 84:11(1981);                                                             Scott et al, Photochem.                                                       Photobiol. 34:63(1981)                                     5-aminomethyl-8-   Hansen et al, Tet. Lett.                                   methoxypsoralen    22:1847(1981)                                              4,5,8-trimethylpsoralen                                                                          Ben-Hur et al,                                                                Biochem. Biophys.                                                             Acta 331:181(1973)                                         4'-aminomethyl-4,5,8-                                                                            Issacs et al, Biochem.                                     trimethylpsoralen  16:1058(1977)                                              xanthotoxin        Hradecma et al, Acta                                                          Virol. (Engl. Ed.) 26:                                                        305(1982)                                                  khellin            Beaumont et al,                                                               Biochim. Biophys.                                                             Acta 608:1829(1980)                                   R.   Benzodipyrones     Murx et al, J. Het.                                                           Chem. 12:417(1975);                                                           Horter et al, Photo-                                                          chem. Photobiol. 20:                                                          407(1974)                                             S.   Monostral Fast Blue                                                                              Juarranz et al, Acta                                                          Histochem. 70:130 (1982)                              ______________________________________                                    

Particularly useful photoreactive forms of such intercalating agents arethe azidointercalators. Their reactive nitrenes are readily generated atlong wavelength ultraviolet or visible light and the nitrenes ofarylazides prefer insertion reactions over their rearrangement products[see White et al, Methods in Enzymol. 46:644(1977)]. Representativeazidointercalators are 3-azidoacridine, 9-azidoacridine, ethidiummonoazide, ethidium diazide, ethidium dimer azide [Mitchell et al, JACS104:4265(1982)], 4-azido-7-chloroquinoline, and 2-azidofluorene. Otheruseful photoreactable intercalators are the furocoumarins which form[2+2] cycloadducts with pyrimidine residues. Alkylating agents can alsobe used such as bis-chloroethylamines and epoxides or aziridines, e.g.,aflatoxins, polycyclic hydrocarbon epoxides, mitomycin, and norphillinA.

The label which is linked to the nucleic acid component according to thepresent invention can be any chemical group or residue having adetectable physical or chemical property. The label will bear afunctional chemical group to enable it to be chemically linked to theintercalator compound. Such labeling materials have been well developedin the field of immunoassays and in general most any label useful insuch methods can be applied to the present invention. Particularlyuseful are enzymatically active groups, such as enzymes (see Clin. Chem.(1976)22:1243), enzyme substrates (see British Pat. Spec. No.1,548,741), coenzymes (see U.S. Pat. Nos. 4,230,797 and 4,238,565), andenzyme inhibitors (see U.S. Pat. No. 4,134,792; fluorescers (see Clin.Chem. (1979)25:353) and chromophores including phycobiliproteins;luminescers such as chemiluminescers and bioluminescers (see Clin. Chem.(1979)25:512, and ibid, 1531); specifically bindable ligands; andresidues comprising radioisotopes such as ³ H, ³⁵ S, ³² P, ¹²⁵ I, and ¹⁴C. Such labels are detected on the basis of their own physicalproperties (e.g., fluorescers, chromophores and radioisotopes) or theirreactive or binding properties (e.g., enzymes, substrates, coenzymes andinhibitors). For example, a cofactor-labeled nucleic acid can bedetected by adding the enzme for which the label is a cofactor and asubstrate for the enzyme. A hapten or ligand (e.g., biotin) labelednucleic acid can be detected by adding an antibody or an antibodyfragment to the hapten or a protein (e.g., avidin) which binds theligand, tagged with a detectable molecule. Such detectable molecule canbe some molecule with a measurable physical property (e.g., fluorescenceor absorbance) or a participant in an enzyme reaction (e.g., see abovelist). For example, one can use an enzyme which acts upon a substrate togenerate a product with a measurable physical property. Examples of thelatter include, but are not limited to, β-galactosidase, alkalinephosphatase, papain, and peroxidase. For in situ hybridization studies,ideally the final product is water insoluble. Other labels will beevident to one of the ordinary skill in the art.

The label will be linked to the intercalator compound by direct chemicallinkage such as involving covalent bonds, or by indirect linkage such asby the incorporation of the label in a microcapsule or liposome which inturn is linked to the intercalator compound. Methods by which the labelis linked to the intercalator compound are essentially known in the artand any covenient method can be used to perform the present invention.

Advantageously the intercalator compound is first combined with thelabel chemically and thereafter combined with the nucleic acidcomponent. For example, since biotin carries a carboxyl group it can becombined with a furocoumarin by way of amide or ester formation withoutinterfering with the photochemical reactivity of the furocoumarin or thebiological activity of the biotin, e.g., ##STR2## Otheraminomethylangelicin, psoralen and phenanthridium derivatives can besimilarly reacted, as can phenanthridium halides and derivatives thereofsuch as aminopropyl methidium chloride, i.e. ##STR3## [see Hertzberg etal, J. Amer. Chem. Soc. 104:313(1982)].

Alternatively a bifunctional reagent such as dithiobis succinimidylpropionate or 1,4-butanediol diglycidyl ether can be used directly tocouple the photochemically reactive molecule with the label where thereactants have alkyl amino residues, again in a known manner with regardto solvents, proportions and reaction conditions. Certain bifunctionalreagents, possibly glutaraldehyde may not be suitable because, whilethey couple, they may modify the nucleic acid and thus interfere withthe assay. Routine precautions can be taken to prevent suchdifficulties.

The particular sequence in making the labeled nucleic acid can bevaried. Thus, for example, an amino-substituted psoralen can first bephotometrically coupled with a nucleic acid, the product having pendantamino groups by which it can be coupled to the label. Alternatively, thepsoralen can first be coupled to a label such as an enzyme and then tothe nucleic acid.

The spacer chain length between the nucleic acid-binding ligand and thelabel can be extended via hydrocarbon or peptide. A typical exampleinvolves extending an 8-hydroxy psoralen derivative with an alkylhalide, according to the method described by J. L. DeCout and J. Lhomme,Photochemistry Photobiology, 37, 155-161 (1983). The haloalkylatedderivative is then reacted either with thiol or amines to produce thereactive residue, as has been described by W. A. Saffran et al., Proc.Natl. Acad. Sci., U.S.A., 79, 4594 (1982)

If the label is an enzyme, for example, the product will ultimately beplaced on a suitable medium and the extent of catalysis will bedetermined. Thus, if the enzyme is a phosphatase the medium couldcontain nitrophenyl phosphate and one would monitor the amount ofnitrophenol generated by observing the color. If the enzyme is aβ-galactosidase the medium can containo-nitrophenyl-D-galacto-pyranoside which also will liberate nitrophenol.

The labeled nucleic acid of the present invention is applicable to allconventional hybridization assay formats, and in general to any formatthat is possible based on formation of a hybridization product oraggregate comprising the labeled nucleic acid. In particular, the uniquelabeled probe of the present invention can be used in solution andsolid-phase hybridization formats, including, in the latter case,formats involving immobilization of either sample or probe nucleic acidsand sandwich formats.

The labeled nucleic acid probe will comprise at least one singlestranded base sequence substantially complementary to or homologous withthe sequence to be detected. However, such base sequence need not be asingle continuous polynucleotide segment, but can be comprised of two ormore individual seqments interrupted by nonhomologous sequences. Thesenonhomologous sequences can be linear or they can be self-complementaryand form hairpin loops. In addition, the homologous region of the probecan be flanked at the 3'- and 5'-terminii by nonhomologous sequences,such as those comprising the DNA or RNA of a vector into which thehomologous sequence had been inserted for propagation. In eitherinstance, the probe as presented as an analytical reagent will exhibitdetectable hybridization at one or more points with sample nculeic acidsof interest. Linear or circular single stranded polynucleotides can beused as the probe element, with major or minor portions being duplexedwith a complementary polynucleotide strand or strands, provided that thecritical homologous segment or segments are in single stranded form andavailable for hybridization with sample DNA or RNA. Useful probesinclude linear or circular probes wherein the homologous probe sequenceis in essentially only single stranded form [see particularly, Hu andMessing, Gene 17:271(1982)].

The labeled probe of the present invention can be used in anyconventional hybridization technique. As improvements are made and asconceptually new formats are developed, such can be readily applied tothe present labeled probe. Conventional hybridization formats which areparticularly useful include those wherein the sample nucleic acids orthe polynucleotide probe is immobilized on a solid support (solid-phasehybridization) and those wherein the polynucleotide species are all insolution (solution hybridization).

In solid-phase hybridization formats, one of the polynucleotide speciesparticipating in hybridization is fixed in an appropriate manner in itssingle stranded form to a solid support. Useful solid supports are wellknown in the art and include those which bind nucleic acids eithercovalently or non-covalently. Noncovalent supports which are generallyunderstood to involve hydrophobic bonding include naturally occurringand synthetic polymeric materials, such as nitrocellulose, derivatizednylon, and fluorinated polyhydrocarbons, in a variety of forms such asflters or solid sheets. Covalent binding supports are also useful andcomprise materials having chemically reactive groups or groups, such asdichlorotriazine, diazobenzyloxymethyl, and the like, which can beactivated for binding to polynucleotides.

A typical solid-phase hybridization technique begins with immobilizationof sample nucleic acids onto the support in single stranded form. Thisinitial step essentially prevents reannealing of complementary strandsfrom the sample and can be used as a means for concentrating samplematerial on the support for enhanced detectability. The polynucleotideprobe is then contacted with the support and hybridization detected bymeasurement of the label as described herein. The solid support providesa convenient means for separating labeled probe which has hybridized tothe sequence to be detected from that which has not hybridized.

Another method of interest is the sandwich hybridization techniquewherein one of two mutually exclusive fragments of the homologoussequence of the probe is immobilized and the other is labelled. Thepresence of the polynucleotide sequence of interest results in dualhybridization to the immobilized and labeled probe segments See Methodsin Enzymology 65:468(1980) and Gene 21:77-85(1983) for further details.

The invention will be further described in the following exampleswherein parts are by weight unless otherwise expressed.

EXAMPLE 1

50 mg of N-hydroxysuccinimido biotin is dissolved in 2 mldimethylsulfoxide (soln A). 10 mg of 4' aminomethyl trioxsalen(structure 1) (or other aminoalkyl compounds) is dissolved in 10 ml(soln B) aqueous buffer (e.g., 10 mM sodium tetraborate, pH adjustedwith HCl) solution pH˜8. Solution (A) and (B) are mixed in a volumeratio of 1:10 and weight ratio of 10:1, so that the activated hapten ispresent in large excess. The reaction is allowed to proceed at 35° C.for 1 hour. The extent of the reaction is monitored by thin layerchromatography--on silica gel plates with a fluorescence indicators in asolvent 1/1/8--methanol/Acetic acid/chloroform. Under these TLCconditions unreacted aminomethyl trioxalane moves with the solvent frontwhereas the product has a slower mobility. Biotin does not show anyfluorescence but the adduct fluoreces because of trioxsalen. Growth ofthe new fluorescent spot and disappearance of the original fluorescentspot indicates the extent of product formation. Since the activatedbiotin is in large excess, fluorescence corresponding to the startingmaterial vanishes on TLC after the completion of reaction. Excess activebiotin is reacted with glycyl-glycine or lysine. The presence of aminoacid biotin product does not interfere with the photochemical reactionof psoralen-biotin compounds with DNA. Hence, a purification step afterthe above reaction is not essential.

EXAMPLE 2

100 mg of biotin nitrophenyl ester is dissolved in dry DMSO (2-5 ml) and10 mg of 4'-aminomethyl trioxsalen is dissolved in dry DMSO (5 ml). Thetwo solutions are mixed in a molar ratio so that biotin nitrophenylester is about ten times with respect to 4'-aminomethyl trioxsalen. 100ml of triethylamine is added to the mixture and shaken well. Theprogress of reaction is checked by TLC and excess unreacted biotinnitrophenyl ester is reacted with lysine as in Example 1. The reactionis allowed to proceed for 1 hour at 35° C. and then lysine is added toquench the reaction. After the reaction, DMSO is evaporated under vacuumand the gummy residue is taken in methanol and can bechromatographically purified on an LH 20 column, using methanol as aneluant. The last step is not essential for the photochemical interactionof psoralen adduct with DNA.

EXAMPLE 3

Biotin can be coupled to aminoalkyl hydroxyalkyl compounds bycarbodiimide mediated reaction. 10 mg biotin is dissolved in 1 mldimethyl formamide. To the solution, 5 mg of 4'-hydroxymethyl trioxsalenis added followed by 10 mg dicyclohexyl carbodiimide. The reaction isallowed to proceed for 20 hours at room temperature, dicyclohexylureaprecipitate is removed and the product is recovered by removing DMFunder vacuum. The same reaction can be performed in pyridine.

The foregoing examples will be give similar results if the aminoalkyltrioxsalen is replaced by other aminoalkyl furocoumarins, phenanthridiumhalides, and the like.

EXAMPLE 4 Coupling of an enzyme to a photoactive amino compound and thencovalent attachment to DNA

A typical examle is given with papain. 0.1 mg/ml of papain solution in100 mM phosphate buffer (pH 8) is added to 10 mg/ml of amino methyltrioxsalen. The final solution should be 1:1 with respect to volume ofenzyme and photoactivator solution. Then solid dithiobis-succinimidylpropionate or dimethyl suberimidate is added to a final concentration of20 μg/ml. The pH is continuously monitored and maintained at theoriginal value by 0.001M sodium hydroxide. After adding the crosslinkertwice, the reaction is allowed to proceed for 30 minutes at roomtemperature. The free photoactive amine is separated from theenzyme-bound compounds by gel filtration on Sephadex G-10. The adduct isexcluded along with the free protein and protein-protein conjugates.Most of these impurities have very little effect on DNA binding. Anyenzyme which has been modified and still retains its activity can becoupled similarly.

After the purification, the enzyme conjugate is mixed with DNA inaqueous buffer (pH 7.5) and irradiated at 390 nm for 1 hour. The adductis separated from the unreacted residues on Sephadex (G-100) column. Theactivity is tested as follows: DNA-enzyme conjugate is dialyzed against10 mM EDTA-containing buffer (pH 6.2). To 8 ml of the DNA-enzymesolution, 10 ml of 60 mM mercaptoethanol and 1 ml 50 m mol cysteine(freshly prepared) are added. This is treated as enzyme solution. Thesubstrate solution is prepared as follows:

592 mg benzoyl-L-arginine ethyl ester hydrochloride is dissolved in 30ml water (BAEE).

To this BAEE solution, 1.6 ml 0.01M EDTA, 1.6 ml 0.05M cysteine, freshlyprepared are added, pH is adjusted at 6.2 and the final volume is madeup to 42 ml.

Procedure

Using a pH meter, the following test system has been set up at 25° C.:

5 ml substrate

5 ml H₂ O

5 ml 3M NaCl

1 ml enzyme dilution

The amount of 0.01M NaOH in ml required to maintain a pH of 6.2 isrecorded. A five-minute period is generally satisfactory.

Since enzyme are not stable at higher temperatures, if the conjugatesare used for hybridization assays, low temperatures should be used.(Either oligonucleotides, or an ionic strength less than 2 m molarshould be utilized so that hybridization can be effected at lowtemperature.)

EXAMPLE 5

Identical products are generated if aminoalkylphotoactive compounds arephotoreacted with DNA first, then with the proteins or enzymes orhaptens. DNA (1 mg/ml) and amino methyl trioxsalen (0.1 mg/ml) are mixedin aqueous buffer pH(7.5), and photoirradiated at 390 nm for 1 hour; theproduct is precipitated with ethanol then redissolved in crosslinkingbuffer as in Example 4, and the rest of the procedure is similar.

If monoadduct formation is essential, monoazidoaminopropyl methidium oraminomethyl angelicin compounds are used under otherwise identicalconditions.

EXAMPLE 6

A glycoprotein can be coupled by redox reaction to an aliphatic amine. Atypical example is given below with horse radish peroxidase (HRPO)coupling to 4' aminomethyl trioxsalen. Identical conditions can befollowed with any aminoalkyl compound.

Scheme: ##STR4##

Experiment:

10 mg HRPO (Sigma Chemical Co.) is dissolved in 2 ml freshly prepared0.3M sodium bicarbonate (pH 8.1). To the enzyme solution, 200 microliter1% 2,4-dinitrofluorobenzene in ethanol is added to block α- and ε-aminogroups and some hydroxy groups of the enzyme. The mixture is gentlyshaken for one hour at room temperature. Then 2 ml 80 m molar sodiumperiodate in distilled water is added and mixed for 30 minutes at roomtemperature. In order to quench the unreacted periodate, ethylene glycolis added to a final concentration of 50 m Mol. The solution is dialyzedagainst 10 m molar sodium carbonate buffer (pH 9.5) in a cold room (˜4°C.). To the dialyzed solution, ˜1 mg solid aminomethyl trioxsalen isadded and the mixture is shaken gently for 1 hour at 25° C. 10 mg sodiumborohydride (NaBH₄) solid is added and the reaction is allowed toproceed for 12 hours at 4° C. The adduct is dialyzed against the DNAbinding buffer and then photoreacted by mixing in 1:1 weight ratio(enzyme to DNA) as described before. The separation of the DNA-enzymeadduct from the enzyme is done by gel filtration on a Sephadex G-100column where the adduct is excluded.

To improve the photochemical efficiency, blocking of reactive HRPO sitesbefore oxidation with periodate may be done with allylisothiocyanate, ashas been described by P. K. Nakane et al, Enzyme Labeled Antibodies forLight and Electron Microscopic Localization of Antigens, J. HistochemCytochem, 14, 790 (1966).

Unless stated otherwise, all the reactions are performed in the dark orred light conditions are maintained.

The peroxidase activity is measured by the following method:

100-500 microliters of the sample are mixed with 3 ml 14 mM para-Cresolin 50 m molar tris HCl buffer (pH 7.5). To this 1 ml 1% H₂ O₂ is added.After 2 minutes, 3 ml 5 m molar sodium cyanides in water are added toquench the reaction. The fluorescence of the solution is measured atexcitation 320 nm, emission 410 nm. H. Perschke and E. Broda, Nature190, 257 (1961); M. Roth, Methods of Biochemical Analysis, vol. 17, ed.D. Glick, Interscience Publisher, N.Y., 1969 P. 236.

EXAMPLE 7 Assay for the label after DNA-DNA hybridization

An illustrative example with a single stage DNA-DNA hybridization ispresented here. The procedure used in the case of two-stagehybridization (Application Ser. No. 511,063, filed July 5, 1983, nowabandoned, refiled as a continuation, application Ser. No. 815,694, nowpending) can also be followed. In application Ser. No. 511,063 there isdisclosed a method for determining whether the nucleic acid in a testsample includes a particular nucleic acid sequence, comprising the stepsof:

(a) extracting the nucleic acids from a test sample,

(b) digesting the extracted nucleic acids with a restriction enzymethereby to effect restriction or not to effect restriction, depending onwhether or not the restriction enzyme recognition site is preciselypresent in a sequence in the test DNA,

(c) treating the product of (b) to form single-stranded nucleic acids,

(d) contacting the single-stranded nucleic acids produced in (c) withfirst and second polynucleotide probes which are complementary torespective first and second portions of the sequence to be detected, thetwo portions being non-overlapping and immediately adjacent to therestriction site in question, and such contact being performed underconditions favorable to hybridization of said first and second probes tothe sequence to be detected, hybridization with both of the probes to atest molecule being dependent upon whether in step (b) restriction didnot occur, the first probe being incorporated with a distinguishablelabel,

(e) separating, by means of the second probe, (i) any resulting dualhybridization product comprising the sequence to be detected hybridizedto both the labeled first probe and the second probe, from (ii) anyunhybridized and singly hybridized labeled first probe, and

(f) by means of said label detecting any of the separated dualhybridization product which may be present. Plasmid pBR322 (New EnglandBiolab) is digested with the restriction endonuclease, Pst 1 and Pvu 1.This double digestion produces one fragment of 126 base pair long DNAcontaining the part of ampicillin resistance gene and another fragmentof 4236 base pair long DNA. The 126 bp long fragment is isolated byrunning the double digest on 5% polyacrylamide gel. A part of this DNAis labeled either with biotin or with enzymes as described before andused as the labeled probe. For hybridization, Pst 1 cut pBR322 (forcontrol) or the test sample DNA is covalently linked to cellulose byphotochemical method (application Ser. No. 511,064, filed July 5, 1983now U.S. Pat. No. 4,542,102 issued Sept. 17, 1985), by cyanogen bromideactivation or by diazotization method (H. Bunemann, Nucleic Acids Res.,10, 7181 (1982)).

The cellulose containing the denatured DNA is suspended in 5 m molarsalt solution for hybridization with enzyme-coupled DNA or suspended in2.4M tetraethylammonium chloride when biotinylated DNA is used. Thenhybridization is done as described by H. Bunemann in Nucleic AcidsResearch, 10, 7181 (1982) for the detection of the ampicillin resistancegene using 126 base pairs labeled fragment as the probe. In low salt,hybridization is done at 30°-40° C.; in 2.4M (high salt), it is donebetween 40° and 50° C.

After hybridization, FITC-labelled avidine is used to assay for biotinor proper enzyme assay is done with the particles.

It will be understood that the specification and examples areillustrative but not limitative of the present invention and that otherembodiments within the spirit and scope of the invention will suggestthemselves to those skilled in the art.

What is claimed is:
 1. A hybridizable labeled nucleic acid comprising(a) a nucleic acid component, (b) a mono-adduct forming nucleicacid-binding ligand photochemically linked to the nucleic acid componentthereby modifying the nucleic acid such that the frequency ofmodification along a hybridizable single stranded portion of the nucleicacid not be so great as to substantially prevent hybridization andwherein the nucleic acid is modified at not more than one site per 25nucleotide bases, and (c) a label chemically linked to (b).
 2. Ahybridizable labeled nucleic acid according to claim 1, wherein thenucleic acid-binding ligand is an intercalator compound selected fromthe group consisting of acridine dyes, phenanthridines, phenazines,furocoumarins, phenothiazines and quinolines.
 3. A hybridizable labelednucleic acid according to claim 2, wherein the intercalator compound isa furocoumarin or a phenanthridine.
 4. A hybridizable labeled nucleicacid according to claim 1, wherein the label (c) is a specificallybindable ligand.
 5. A hybridizable labeled nucleic acid according toclaim 1, wherein the label is selected from the group consisting of ahapten, biotin, an enzyme, a fluorescent radical and a luminescentradical.
 6. A hybridizable labeled nucleic acid according to claim 5,wherein the fluorescent radical is from fluorescein.
 7. A hybridizablelabeled nucleic acid according to claim 1, wherein the label (c) is aphycobiliprotein.
 8. A hybridizable labeled nucleic acid according toclaim 5, wherein the chemical link between the biotin and (b) iseffected via succinimidyl activation.
 9. A hybridizable labeled nucleicacid according to claim 5, wherein (b) is the radical of anamino-substituted angelicin or psoralen and is linked to the biotinthrough an amide group.
 10. A hybridizable labeled nucleic acidaccording to claim 5, wherein the enzyme is selected from the groupconsisting of β-galactosidase, horse radish peroxidase and papain.
 11. Ahybridizable labeled nucleic acid according to claim 1, wherein thenucleic acid component is in single stranded form.
 12. A method ofmaking a hybridizable labeled nucleic acid, which comprises contacting anucleic acid with an adduct, said adduct being suitable forphotochemical attachment to a nucleic acid probe, said adduct comprisinga mono-adduct forming nucleic acid-binding ligand and a label chemicallylinked thereto, and subjecting the nucleic acid and adduct tophotochemical irradiation thereby modifying the nucleic acid such thatthe frequency of modification along a hybridizable single strandedportion of the nucleic acid not be so great as to substantially preventhybridization and wherein the nucleic acid is modified at no more thanone site per 25 nucleotide bases.
 13. A labeled hybridizable nucleicacid comprising(a) a nucleic acid component, (b) a mono-adduct formingintercalator compound photochemically linked to the nucleic acidcomponent thereby modifying the nucleic acid such that the frequency ofmodification along a hybridizable single stranded portion of the nucleicacid not be so great as to substantially prevent hybridization andwherein the nucleic acid is modified at not more than one site per 25nucleotide bases, and (c) a label chemically linked to (b), the labeledhybridizable nucleic acid produced by(i) photochemically linking thenucleic acid component to the intercalator compound and chemicallylinking a label to the photochemically linked intercalator compound or(ii) chemically linking a label to the intercalator compound to form acomposite and photochemically linking said composite to the nucleic acidcomponent.
 14. A labeled hybridizable nucleic acid comprising(a) anucleic acid component, (b) a mono-adduct forming nucleic acid bindingligand photochemically linked to the nucleic acid component therebymodifying the nucleic acid such that the frequency of modification alonga hybridizable single stranded portion of the nucleic acid not be sogreat as to substantially prevent hybridization and wherein the nucleicacid is modified at not more than one site per 25 nucleotide bases, (c)a linker group coupled to the nucleic acid binding ligand and (d) alabel chemically linked through the linker group to the nucleic acidbinding ligand.